G6PI介导RA关节滑膜增生与血管新生的作用及分子机制

Glucose-6 - phosphate isomerase (G6PI) is a ubiquitous cytosolic enzyme that catalyzes the second step in glycolysis. Molecular cloning and sequencing have identified G6PI as autocrine motility factor (AMF), also known as neuroleukin (NLK). AMF is a multifunctional cytokine that exhibits multifunctional growth factor-like activity via a unique cognate 78 kDa (gp78) seven-transmembrane glycoprotein receptor (autocrine motility factor receptor, AMFR). Overexpression of AMF and AMFR has been found in a wide spectrum of malignancies and is associated with cancer progression, metastasis, and angiogenesis. It has been reported that G6PI had the capacity to induce the chronic arthritis in mice. Our previous study found that the concentrations of soluble G6PI in the sera and synovial fluids of RA patients were elevated, and the sera GPI level correlated positively with the disease activity of RA. G6PI also enhanced the proliferation capacity and prohibit apoptosis on cultured RA-FLS. But so far it is remains unclear that pathogenic role. This study will continue to explore the following research on the basis of preliminary work: 1. To clarify the characteristic of G6PI /AMFR expression and autocrine G6PI on RA-FLS. 2. Construction the vectors of G6PI expression and siRNA interference, to investigate the changes of FLS on proliferation capacity and secretion of inflammatory cytokines after the G6PI expression is interference. 3. To explore the roles and its molecular mechanism of G6PI/AMFR pathway on FLS proliferation capacity and synovial angiogenesis by means of G6PI inhibitor, G6PI, anti-G6PI antibodies, anti-AMFR antibodies and AMFR-Fc fusion protein. 4. Using a variety of ways, including in vitro angiogenesis assay, in vivo matrigel plug assay and the mouse RA model, to verification the role of G6PI /AMFR pathway in induced synovial hyperplasia and angiogenesis.

葡萄糖6磷酸异构酶(G6PI)在细胞内是糖酵解关键酶，在细胞外又称为自分泌蠕动因子(AMF)，能促进肿瘤生长、转移和血管新生，也能诱导鼠发生关节炎。课题组研究发现类风湿关节炎（RA）患者外周血及关节液中G6PI含量异常增高、增高水平与病情活动正相关，并且G6PI能抑制关节滑膜成纤维样细胞(FLSs)凋亡。目前G6PI在RA发病机制中的作用尚未明。本项目继续深入，构建G6PI表达和siRNA干扰载体，从干扰FLSs细胞内G6PI表达和采用G6PI抑制剂、G6PI抗体、AMF受体（AMFR）抗体、AMFR-Fc融合蛋白干扰G6PI/AMFR信号通路两方面，研究G6PI调控RA-FLS增殖、凋亡、分泌炎症因子和促血管生成因子的作用，并采用体外、体内血管形成实验和G6PI诱导的RA模型来探明G6PI促滑膜血管新生作用，以初步阐明G6PI在RA关节滑膜增生、血管翳形成中的作用及分子机制。

本项目以RA关节滑膜成纤维细胞（RA-FLS）、人真皮微血管内皮细胞（HDMECs）为主要研究对象，探讨G6PI调控RA关节滑膜增生及促血管新生的作用和分子机制。主要完成了以下内容：.1. 阐明RA和骨关节炎（OA）滑膜组织和FLS中G6PI及受体表达分布，首次发现FLS存在自分泌G6PI特性，RA-FLS自分泌G6PI量明显高于OA-FLS。.2. 研究G6PI调控RA-FLS增殖、凋亡的作用及分子机制。研究发现FLS细胞内G6PI表达增加，可以显著促进FLS增殖，相反FLS生长缓慢；外源性G6PI刺激，同样促进FLS增殖，G6PI抗体则显著抑制FLS增殖。机制研究显示G6PI与其受体（gp78）结合后，促使受体迅速磷酸化、激活ERK蛋白的级联反应，促进细胞G1→S期转换，从而促进FLS的增殖，同时抑制Fas介导的细胞调亡。另外G6PI可以促进炎症因子IL-1β和TNF-α的分泌。.3. 研究G6PI诱导关节滑膜血管新生的作用及作用机制。低氧环境RA-FLS与HDMECs共培养、促进HDMECs成管，抑制RA-FLS内G6PI表达，则RA-FLS的促进成管作用消失，进一步抑制HDMECs内G6PI表达，其成管能力也显著降低。分析其机制发现，低氧环境RA-FLS、HDMECs表达G6PI、HIF-1α、VEGF均显著增加，G6PI表达受HIF-1α调控，而VEGF表达受HIF-1α、G6PI双调控。此外研究还发现，不管是外部环境还是细胞内G6PI增多，均能促进HDMECs迁移和侵袭。以上结果表明G6PI在缺氧诱导HDMECs迁移、侵袭、新生血管中起着关键的作用。.4. 缺氧环境也诱导FLS表达烯醇化酶1（ENO1）、硫氧还蛋白1（thioredoxin 1, Trx1），本项目在基本完成G6PI相关研究之余，探讨了ENO1、Trx1表达对RA-FLS增殖、凋亡的影响。研究发现在缺氧环境中HIF-1α与Trx1存在相互促进表达的关系并共同促进VEGF过度表达.在FLS中，Trx1通过调控PI3K-Akt信号转导通路促进RA-FLS增殖、抑制凋亡。研究揭示Trx1是个重要的促进RA关节滑膜增生、血管新生的靶分子。此外，缺氧也促进FLS表达ENO1，ENO1可促进FLS增生、抑制凋亡。